Myocardial ischemia/reperfusion (I/R) injury is a major human health problem causing millions of deaths per year. Myocardial I/R involves severe cardiac myocyte dysfunction and myocyte death for which there is no cure. It has been challenging to dissect the role of specific mechanistic pathways in I/R owing to the multitude of cellular processes altered in I/R, including elevated reactive oxygen species (ROS), membrane instability, intracellular Ca2+ mishandling, mitochondrial uncoupling and others. Despite these challenges, myocyte generated ROS is widely considered a key initiating event leading to cellular damage in cardiac I/R. We provide exciting new evidence of a significant uncoupling of myocyte-generated ROS and damage in cardiac I/R. Thus, this proposal focuses on a new paradigm in testing the hypothesis that sarcolemma stability, independent of increased ROS production, is central to I/R injury. To interrogate I/R mechanisms we will implement sarcolemma stabilizers that in preliminary work significantly limit myocyte leak, Ca2+ mishandling, mitochondrial membrane depolarization and preserve myocyte viability despite I/R-mediated increased ROS. These results challenge the dogma of the mechanism of I/R damage by highlighting sarcolemma stabilization as central in the I/R injury pathway. Sarcolemma stabilization by cell surface interacting synthetic copolymers is proposed here as a tool for mechanistic dissection of the direct role of cardiac muscle membrane integrity in I/R. Copolymer-based membrane stabilizers are amphiphilic long-chain macromolecular copolymers that interact with and protect cellular membranes during stress. The overarching hypothesis of this proposal is that, independent of I/R-mediated ROS production, synthetic sarcolemma stabilizers function to significantly limit myocyte Ca2+ dysregulation and mitochondrial depolarization to increase heart pump performance in vivo. Stated differently, we will test the hypothesis that ROS alone is insufficient to cause I/R injury. This hypothesis, if correct, will change the field by providing direct evidence in establishing membrane integrity as central in I/R pathogenesis. The impact of this work derives from using synthetic sarcolemma stabilizers as a viable new therapeutic option that could be readily translated to clinical settings of I/R. The Specific Aims are: Aim 1. To test the hypothesis that during I/R, independent of myocyte ROS production, copolymer sarcolemma stabilizers will significantly limit membrane leak, intracellular Ca2+ mishandling and mitochondrial membrane depolarization to promote cell viability in rodent adult myocytes and human iPSC-derived cardiac myocytes in vitro. Aim 2. To test the hypothesis that copolymer sarcolemma stabilization will promote myocyte viability and preserve myocardial function in a clinically relevant porcine model of myocardial I/R injury in vivo.